Treating disease with resonance generating electromagnetic fields

ABSTRACT

The present disclosure relates, according to some embodiments, to an electromagnetic resonance-based disease treatment system that comprises a processing unit configured to generate a resonant frequency signal and a radiating antenna configured to radiate an electromagnetic field based on the resonant frequency signal. The resonant frequency signal may carry at least one frequency at which reference materials related to a disease condition resonate. An antenna configuration may be used to determine one or more resonant frequencies of the reference materials. A subject having or at risk of having the disease condition may be exposed to the electromagnetic field in order to treat the disease condition.

TECHNICAL FIELD

The present disclosure relates, in some embodiments, to treat humanand/or non-human animal disease conditions involving resonance-basedelectromagnetic radiation.

BACKGROUND

Glioblastoma multiforme (GBM) is an aggressive primary tumour thatarises in the glial cells of the brain, and accounts for approximatelyhalf of all brain tumours. GBM is relatively rare with an incidence ofapproximately three cases per 100,000 person-years. However, theaggressive nature of the disease and the limited treatment optionscombine to negatively impact overall survival. Current standardizedfirst-line treatment for GBM may comprise surgical resection(debulking), followed by six weeks of radiotherapy and concurrentchemotherapy with an alkylating agent, temozolomide. Even with thisaggressive regimen, which is associated with considerable morbidity,median survival time after a standard primary intervention is less thana year, with less than 5% of patients surviving for five years. Sincemany GBM tumours are resistant to temozolomide due to methylation by theO6-methylguanine DNA methyl transferase (MGMT) gene, there has been acontinuing search for new chemotherapeutic agents that can induceapoptosis in GBM tumours, particularly for second-line treatments.Example efforts include intralesional implantation of carmustine wafers,antiangiogenic therapies with a humanized monoclonal antibody targetingthe HGF/SF:cMet axis, and methods using low intensity, intermediatefrequency alternating electric fields (TTFields) to induce cell cyclearrest and cell death in tumours.

SUMMARY

A need has arisen for improved approaches to treating conditions such asGBM by using non-invasive modulated electromagnetic radiations thatavoids cumbersome calculations and trial-and-error tests.

The present disclosure relates, according to some embodiments, tomethods of administering to a subject having or at risk of having adisease condition with resonance-based electromagnetic radiation. Forexample, a method may comprise selecting one or more reference materialsrelated to the disease condition; capturing one or more resonantfrequencies of the reference materials; generating a resonant frequencysignal comprising at least one of the captured resonant frequencies;radiating an electromagnetic field based on the resonant frequencysignal; and exposing the subject to the electromagnetic field to affectbiological activities of cells related to the disease condition.

In some embodiments, a method may comprise generating a modulatedbroadband signal that is simultaneously modulated in a signal generatorby a first waveform at a first frequency and at least a second waveformat a second frequency. An electromagnetic field may be effectuated basedon both a resonant frequency signal and a modulated broadband signal.

In some embodiments, a method may comprise generating, by a signalgenerator, a booster signal. The booster signal may comprise a single,low frequency waveform having a frequency lower than 100 KHz. Anelectromagnetic field may be effectuated further based on the boostersignal. In some embodiments, the first frequency of the first waveformmay be substantially identical with the low frequency of the boostersignal but different from the second frequency of the second waveform.In some embodiments, the modulated broadband signal has a bandwidth of 1MHz or greater, and the first frequency of the first waveform is about 1Hz or about 4 Hz, whereas the second frequency of the second waveform isless than 1 MHz.

In some embodiments, a method may further comprise selecting one or moresecond reference materials related to the disease condition; capturingone or more second resonant frequencies of the second referencematerials; generating a second resonant frequency signal comprising atleast one of the captured second resonant frequencies; radiating asecond electromagnetic field based on the second resonant frequencysignal; and exposing the subject to the second electromagnetic fieldsimultaneously with exposure to the electromagnetic field.

In some embodiments, a method may comprise tuning frequencies of theelectromagnetic field via a feedback loop to maximize resonance of thereference materials.

In some embodiments, reference materials may be critical to thefunction, progression, viability, and/or continuation of a cell ororganism causing or associated with a disease condition. Sometimes,after selecting reference materials but before capturing one or moreresonant frequencies, reference materials may be placed and isolatedinside a shielded container. In some embodiments, a resonant frequencysignal comprises at least two captured resonant frequencies, includingany harmonics that are used simultaneously to expose the subject.

In some embodiments, a modulated broadband signal may be received by afirst broadband antenna via cable, and a booster signal and a resonantfrequency signal may be received by a second broadband antenna viacables. An electromagnetic field used to expose the subject may beeffectuated based on radiations from both the first and second broadbandantennae, which are coupled in an antenna box. In some embodiments, theexposure of the subject to the electromagnetic field may last for asufficient cumulative period of time to cause death of cells related tothe disease condition or slow the progression of the disease condition.

In some embodiments, a disease condition may relate to GlioblastomaMultiforme (GBM). Here reference materials may be selected from a group,for example, of hsa-miRNA-38, mutated alpha-kinase 2 gene, Hsp70 (70 kDaheat shock protein), CHI3L1 (chitinase-3-like protein 1), and GBM cells.

In some embodiments, a disease condition may relate to MycobacteriumTuberculosis (Mtb). Here reference materials may be selected from agroup, for example, of Phosphatidylmyo-inositol Mannosides (PIM),Arabinogalactan, Lipoarabinomannan (LAM), and Alpha-crystallin.

In some embodiments, a disease condition may relate to HumanImmunodeficiency Virus (HIV). Here reference materials may be selectedfrom a group, for example, of gp120, gp41, gp160, Gag polyprotein, Envprotein, sequences of viral RNA, p24 protein, and pro-viral DNA.

The present disclosure relates, in some embodiments, to anelectromagnetic resonance-based disease treatment system. A system maycomprise a processing unit configured to generate a resonant frequencysignal that carries at least one frequency at which reference materialsrelated to a disease condition resonate; and a radiating antennaconfigured to radiate an electromagnetic field based on the resonantfrequency signal. An electromagnetic field may be operable on a subjecthaving or at risk of having the disease condition by exposing thesubject to the electromagnetic field in order to treat the diseasecondition.

In some embodiments, a processing unit may comprise a signal generatorconfigured to generate a modulated broadband signal at least viasimultaneously modulation by a first waveform at a first frequency andat least a second waveform at a second frequency. An electromagneticfield may be effectuated based on both the resonant frequency signal andthe modulated broadband signal.

In some embodiments, a signal generator is further configured togenerate a booster signal comprising a single, low frequency waveformbelow about 100 KHz. An electromagnetic field may be further based onthe booster signal. In some embodiments, the first frequency of thefirst waveform is substantially identical with the low frequency of thebooster signal but different from the second frequency of the secondwaveform. In some embodiments, a resonant frequency signal may carry aplurality of resonant frequencies that are used simultaneously to exposethe subject.

In some embodiments, a processing unit may comprise a multi-frequencysignal generator or multiple single frequency signal generatorsconfigured to generate a plurality of resonant frequencies based oncalculated, digitized, or measured resonant frequencies of the referencematerials.

In some embodiments, a system may comprise a feedback antenna located ina near field zone of the subject and an amplifier coupled to thefeedback antenna and a radiating antenna. A feedback antenna, anamplifier, and a radiating antenna may form a feedback loop operable totune frequencies of the electromagnetic field.

The present disclosure relates, in some embodiments, to an apparatus foradministering to a subject a resonance-based electromagnetic radiation.The apparatus may comprise a container configured to hold a one or moretarget substances related to a disease condition and an antennaconfiguration configured to determine at least one resonant frequency atwhich the target substances resonate.

In some embodiments, an apparatus may comprise a processing unit coupledto an antenna configuration and configured to generate a modulatedbroadband signal that is simultaneously modulated at two frequencies oftwo different waveforms and a resonant frequency signal encompassing theat least one resonant frequency. An apparatus may further comprise aradiating antenna configured to radiate a modulated electromagneticfield based on the modulated broadband signal and the resonant frequencysignal, wherein the modulated electromagnetic field is operable toexpose the subject.

The present disclosure relates, in some embodiments, to a system foradministering to a subject a resonance-based electromagnetic radiation.The system may comprise means for selecting one or more targetsubstances relevant to a disease condition and means for determining atleast one resonant frequency at which the target substances resonate.

In some embodiments, a system may comprise means for generating abroadband signal that is simultaneously modulated at two frequencies oftwo different waveforms and a resonant frequency signal comprising theat least one resonant frequency; means for radiating an electromagneticfield based on the broadband signal and the resonant frequency signal;and means for exposing a subject carrying the disease condition to theelectromagnetic field to affect biological activities of cells relevantto the disease condition.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

Some embodiments of the disclosure may be understood by referring, inpart, to the present disclosure and the accompanying drawings, wherein:

FIG. 1 illustrates a disease treatment system that may transmit aresonant frequency according to a specific example embodiment of thedisclosure;

FIG. 2 illustrates a disease treatment system that may transmit multipleresonant frequencies according to an example embodiment of thedisclosure;

FIG. 3 illustrates a disease treatment system (comprising an amplifier)that may transmit a resonant frequency according to a specific exampleembodiment of the disclosure;

FIG. 4 illustrates a disease treatment system that may transmit aresonant frequency without a reference material according to a specificexample embodiment of the disclosure;

FIG. 5 illustrates a disease treatment system (comprising an amplifier)that may transmit multiple resonant frequencies according to an exampleembodiment of the disclosure;

FIG. 6 illustrates a process for treating a disease condition withtargeted electromagnetic radiation according to an example embodiment;

FIGS. 7A-7B illustrate example frequency spectra according to exampleembodiments;

FIG. 7A illustrates an example frequency spectrum captured usingBacillus Calmette-Guerin (BCG), an attenuated strain of Mycobacteriumbovis, as a reference material;

FIG. 7B illustrates another example frequency spectrum captured usingreference materials related to Glioblastoma Multiforme (GBM).

FIG. 7B-1 illustrates, individually, the first of five traces shown inFIG. 7B which represents the spectral difference between cycle 7 and theaverage of five cycles taken at the 2 hour point.

FIG. 7B-2 illustrates, individually, the second of five traces shown inFIG. 7B which represents the spectral difference between cycle 6 and theaverage of five cycles taken at the 2 hour point.

FIG. 7B-3 illustrates, individually, the third of five traces shown inFIG. 7B which represents the spectral difference between the average offive cycles taken at the 2 hour point and itself.

FIG. 7B-4 illustrates, individually, the fourth of five traces shown inFIG. 7B which represents the spectral difference between cycle 5 and theaverage of five cycles taken at the 2 hour point.

FIG. 7B-5 illustrates, individually, the fifth of five traces shown inFIG. 7B which represents the spectral difference between cycle 4 and theaverage of five cycles taken at the 2 hour point.

FIG. 8 illustrates an example experimental setup according to an exampleembodiment;

FIG. 9 illustrates an example comparison of three spectral fieldstrengths according to an example embodiment;

FIGS. 10A-10E illustrate comparisons of changes in control cells versusexposed cells at slow modulation for the U-87 MG strain of GBM,according to an example embodiment;

FIG. 10A shows changes in U-87 MG cell number;

FIG. 10B shows changes in U-87 MG cell cycle;

FIG. 10C shows changes in DNA fragmentation in U-87 MG in all fourquadrants of a histogram;

FIG. 10D shows changes in caspase-3/7 activation;

FIG. 10E shows changes in phosphotidylserine (PS) as measured by AnnexinV staining;

FIGS. 11A-11E illustrate an analysis of U-87 MG strain of GBM cellsproliferation and apoptosis after exposure to resonance generatingelectromagnetic fields at fast modulation, according to an exampleembodiment;

FIG. 11A shows changes in U-87 MG cell number;

FIG. 11B shows changes in U-87 MG cell cycle;

FIG. 11C shows changes in DNA fragmentation in U-87 MG in all fourquadrants of a histogram;

FIG. 11D shows changes in caspase-3/7 activation;

FIG. 11E shows changes in PS as measured by Annexin V staining;

FIGS. 12A-12E illustrate an analysis of the U-87 MG strain of GBM cellsproliferation and apoptosis after 72 hours treatment with 25 nMdocetaxel;

FIG. 12A shows changes in U-87 MG cell number;

FIG. 12B shows changes in U-87 MG cell cycle, including the sub G0population;

FIG. 12C shows changes in DNA fragmentation in U-87 MG in all fourquadrants of a histogram;

FIG. 12D shows changes in caspase-3/7 activation;

FIG. 12E shows changes in PS as measured by Annexin V staining; and

FIG. 13 illustrates a change in spectra from cultures exposed for twohours (represented by curve 1310) and the same cultures after beingexposed for 18 hours (represented by curve 1320).

DETAILED DESCRIPTION

The present disclosure relates, in some embodiments, to systems,apparatuses, and methods for treating a condition (e.g., a diseasecondition) by exposing a target (e.g., a DNA sequence, a protein, anenzyme, a mineral etc. within a cell, a tissue, an organ or the body,etc. which is associated with the disease condition) to a modulatedelectromagnetic field including resonant frequencies at which at least aportion of the target resonates. The present disclosure also relates todiscovery of resonant frequencies for any given disease condition.

Substances may exhibit resonance at a fundamental frequency and atspecific harmonic frequencies when exposed to static or time-varyingmagnetic fields. Resonant frequencies for a particular substance mayvary depending on field strength and modulation.

According to some embodiments, determining resonant frequencies does notinvolve cumbersome calculation and trial-and-error to induce resonancein a selected substance. In an example process, first, one or morebiological substances critical to the progression of a particulardisease condition may be selected and isolated. Then, one or moreresonant frequencies of the selected target substance(s) may be measuredand captured by using the substance(s) as a source material or referencematerial (sometimes referred to herein simply as a reference or source).After determination, one or more resonant frequencies may be transmittedby means of broadband transmission. A positive feedback loop may beoptionally employed to amplify and tune a developed externalelectromagnetic field. Further, once an electromagnetic field is tuned,it may be used to expose a subject (e.g., cells, animals, and/orpatients having or at risk of having a target disease such as cancer,virus, bacteria, degenerative condition or other malady). The tunedexternal field may be located within a near field zone of an antennaarray. Therefore, a disease condition may be treated with resonancegenerating electromagnetic fields by targeting substances relevant tothe condition (e.g., key to the function, progression, viability,proliferation, continuation, and/or survival of the disease) to whichthis therapy is applied.

According to some embodiments, multiple reference materials may be usedconcurrently with an appropriate antenna configuration or arrangement.When treating a disease, prolonged exposure of a subject to a tunedelectromagnetic field may induce amplified resonance within targetsubstances, which in turn helps stop a disease. Amplified resonance mayalso be induced in a target substance by simultaneous transmissions ofmultiple single-frequency signals identified and captured by methodsdisclosed herein. A modulated electromagnetic field disclosed herein isnon-invasive and does not require electrodes or other attachments to beconnected to a subject in order to deliver therapy.

FIG. 1 illustrates disease treatment system 100 according to a specificexample embodiment of the disclosure, where a disease may be treatedwith targeted electromagnetic radiation. Disease treatment system 100comprises shielded container 110, broadcast antenna box 130, signalgenerator 150, monitoring antenna 160, and feedback antenna 170,arranged as shown in FIG. 1. Disease treatment system 100 may representa single line broadband setup since one signal line 1 resonant frequencysignal.

As shown in FIG. 1, shielded container 110 may be configured to hold oneor more reference materials (e.g., reference material 112) that arerelevant to a disease condition. A suitable quantity of referencematerial 112 may be extracted from biological cells of a subject thatcarries a disease condition to be treated. This subject may be subject140 to be treated or may be a different subject. In some embodiments,biological and/or other substances critical to the function orprogression of the disease condition are selected to be targeted.Compared to man-made organic materials, reference material 112 may beadvantageous, for example, where resonant frequencies (e.g., exactresonant frequencies) may be determined accurately by testing actualbiological substances of a disease or other substances (e.g., calcium,iron, etc.) key to the function, progression, viability, proliferation,continuation, and/or survival of a disease condition. Further, referencematerial 112 may capture and/or fine-tune resonant frequencies ofbiological and/or other substances with little or no activity as abandpass filter. For example, reference material 112 may act as a tuningfork that absorbs radiated frequencies and resonates at one or morecertain frequencies. Selected substances, such as those in referencematerial 112, may be isolated and placed in a non-conductive vial (e.g.,glass vial). A vial may be seated within a capsule to ensure properpositioning within shielded container 110. Although container 110 isshown as electromagnetically “shielded,” any suitable type of container(e.g., open or closed) may be used as long as the purpose of resonantfrequency determination can be fulfilled.

Shielded container 110 may further comprise (or be coupled to) one ormore of antennae 114, 116, and 118, which are configured to determine orcapture one or more resonant frequencies of reference material 112.Antenna 114 serves as a broadband-compatible driven antenna, whileantenna 116 and 118 serve as broadband-compatible receiving antennae.For instance, reference material 112 may be properly positioned betweendriven antenna 114 and receiving antennae 116 and 118. One or moreresonant frequency signals are captured within a feedback loop as system100 tunes. Signal spectra can be recorded through the use of a spectrumanalyzer, an oscilloscope, and/or proprietary software (e.g., SPAN 32),thus one or more resonant frequencies may be captured within system 100and within the software (e.g., for further use). These various signals,including resonant frequency bands, may be digitized and storedelectronically. Resonant frequency signal 120 may be implemented as anysignal comprising carrying one or more captured resonant frequencies andmay be delivered to broadcast antenna box 130.

As shown in FIG. 1, signal generator 150 may be a multi-channel(broadband and single frequency) signal generator that suppliesmodulated signals to radiating antennae 132 and 134. In an exampleembodiment, signal generator 150 generates two signals, includingmodulated broadband signal 152 and single-frequency modulated signal154, that feed into broadcast antenna box 130. Modulated broadbandsignal 152 may have a bandwidth of one Mega-Hertz (MHz) or greater. Alower-end frequency of modulated broadband signal 152 may be flexiblydetermined to have any suitable value (e.g., with the lowest frequencyno less than 1 Hz, no less than 3 Hz, no less than 10 Hz, or no lessthan 100 Hz). In an example, modulated broadband signal 152 has variousfrequency components between 100-3500 MHz. Modulated broadband signal152 may be simultaneously modulated by at least two different waveforms,each at its own frequency. In an example, modulated broadband signal 152may be simultaneously modulated by a first sinusoidal waveform (e.g.,with a frequency of 1 Hz, 4 Hz, or 16 Hz, etc.) along with modulation bya second waveform at a different frequency than the first modulatingsignal, making signal 152 a double-modulated broadband signal.Simultaneous modulation of a broadband signal 152 by at least twowaveforms occurs inside signal generator 150, and such a design may helpenhance amplified resonance. For example simultaneous modulations via atriangle waveform along with a square waveform may produce increasedamplified resonance. Further, an additional single-frequency modulatedsignal 154 is an optional booster signal, which may be flexiblydetermined to have any suitable value and may be implemented as asingle, lower frequency (e.g., 1 Hz, or 4 Hz, or 16 Hz, or below 1 KHz,or below 100 KHz (such as 2.2 KHz), etc.) waveform without any higherfrequency components. The single, low frequency waveform via separatetransmission may also enhance amplified resonance.

Signals supplied by signal generator 150 may be at very low, low, high,or very high frequencies and may be simultaneously modulated by at leasttwo frequencies (very low, low, high, very high, low and high, or anyother combination thereof) with different and/or similar waveforms. Forinstance, signal generator 150 may supply broadband signal 152—e.g.,with a bandwidth of 1 MHz or greater which is modulated by at least twodifferent waveforms plus an optional booster signal 154 at a single lowfrequency. A first waveform of broadband signal 152 may be substantiallyidentical to booster signal 154 and a second waveform of broadbandsignal 152 may have a different frequency than booster signal 154. Suchwaveform combinations may optimize resonance within the targeted areasof a disease condition.

System 100 may operate with any non-ionizing frequency range and at lowfield or high field emission levels. Low field transmission strength andmodulation typically within radio frequency (RF) to low microwavefrequency range of any suitable frequency end points (e.g., 0 Hz to 6GHz, 100 MHz to 3.5 GHz, or 3 Hz to 10 GHz, etc.) may be used tominimize any potential negative side effects of the therapy. The largerthe bandwidth of transmission, the higher the number of resonantfrequencies (including harmonics) that can be captured and applied fordisease treatment.

As shown in FIG. 1, broadcast antenna box 130 may comprise broadbandantennae 132 and 134. Broadband antenna 132 is configured to receiveresonant frequency signal 120 by cable from broadband antenna 116 and/orbroadband antenna 118. Resonant frequency signal 120 is mixed by cablewith modulated broadband signal 152 (modulated by at least two differentwaveforms at different frequencies simultaneously) and withsingle-frequency modulated booster signal 154, if present, which istransmitted in space both inside and outside broadcast antenna box 130to generate modulated broadband electromagnetic signal 136. Broadbandantennae 132 and 134 radiate signals to form the mixed modulatedbroadband electromagnetic signal 136, which effectuates anelectromagnetic field onto subject 140. Subject 140 may be a patient, ananimal, a cell culture, or any applicable disease carrier. Exposingsubject 140 carrying a disease to the resonance generatingelectromagnetic field would affect biological activities of cells ororganisms (e.g., inhibit progression or growth, or cause death) that arerelevant to the disease. Note that broadcast antenna box 130 may notrequire the use of mixer, amplifier, internal feedback antenna, or astabilizing inner core. Instead, broadcast antenna box 130 may employone or more external feedback antennae, which may not only boost asignal at a distance but also enhance electromagnetic field uniformity.

In some embodiments, disease treatment system 100 may further comprisefeedback antenna 170, which is connected to shielded container 110 toform a feedback loop. Feedback antenna 170 may be a broadband-capableantenna that receives radiated signal 136 and accordingly generatesfeedback signal 172. Therefore, Feedback antenna 170 supplies signal 136back to shielded container 110 to induce feedback resonance withinshielded container 110 and subsequently in radiated antenna(s) 132and/or 134. For example, radiated signal 136 may be supplied to shieldedcontainer 110 by means of a positive feedback loop to induce resonancewithin selected reference material 112. A broadband signal (e.g., signal120) that carries captured resonances from reference material 112 may bethen supplied to radiating antenna 132 as part of the feedback loop.

Both generated electromagnetic signals and signals carrying the specificresonances may be radiated, and such radiation may repeat. Therepetitive feedback action may amplify resonances specific to referencematerial 112 and may quickly cause an external electromagnetic field toreach steady state. For example, as shown in FIG. 1, in an embodimentwith a single reference material and antenna set up, signals fromantennae 132 and 134 may be first radiated as separate signals. Then, asa result of a feedback loop, signals from antennae 132 and 134 may beradiated as mixed signal 136, which is reinforced by received mixedtransmission from the feedback antenna(e) and reinforced by mixing withthe reference/source material signal. An external electromagnetic fieldformed between feedback antenna 170 and radiating antenna 132 may alsobe referred to as near-field zone or area 138 of an antenna array. Afterreaching a steady state, an external electromagnetic field is considered“tuned,” which may be confirmed by use of monitoring antenna 160 and aspectrum analyzer in conjunction with proprietary software (SPAN 32),which records correlation factors between scans. The electromagneticfield may also be monitored by using an oscilloscope. For example,monitoring antenna 160 (e.g., a broadband antenna) may monitor aspectral profile of signal 136 and then send the profile to a spectrumanalyzer for analysis, e.g., in terms of component frequencies andmagnitudes.

As illustrated in FIGS. 2-5, the configuration of FIG. 1 may be alteredin various ways within principle of the present disclosure. One ofordinary skill in the art would recognize similarities between variousembodiments, therefore redundant discussions are omitted. FIG. 2, forexample, illustrates disease treatment system 200 according to anexample embodiment, where multiple resonant frequencies may betransmitted simultaneously to affect a particular disease condition. Forillustration purposes, two sets of shielded containers 110 and111—containing reference materials 112 and 113, respectively, whichgenerate two distinctly different resonant frequency signals 120 and 121respectively—and two sets of broadcast antenna boxes 130 and 131 arearranged in parallel in FIG. 2. Simultaneous processing and transmissionof multiple resonant frequencies may impact target cells moreeffectively with accurate tuning while avoiding unnecessarilyduplicative system components. For example, it is unnecessary to providean additional signal generator or feedback antennae to induce thedesired resonance from multiple signals. Multiple resonant frequenciesmay be identified in parallel through use of multiple source materials.Alternatively, resonant frequencies may be identified via calculation ortrial-and-error. Captured resonant frequencies herein can be storeddigitally or replicated by means of a suitable multi-frequency signalgenerator.

FIG. 3 illustrates disease treatment system 300 according to an exampleembodiment, which is similar to disease treatment system 100 exceptamplifier 171. In a positive feedback loop, amplifier 171 may situatebetween feedback antenna 170 and shielded container 110. Amplifier 171may generate feedback signal 172, which helps amplify resonance thatleads to the disruption of cellular homeostasis and induction of celldeath.

FIG. 4 illustrates disease treatment system 400 according to an exampleembodiment, which removes the need for a reference material in eachsubsequent system used. Instead, resonant frequencies of one or morereference materials may be measured, captured, calculated, or otherwisedetermined off-site or beforehand in accordance with an exampleembodiment, and then recorded manually or digitized. Then, signalgenerator 410 may simultaneously generate digitized, calculated,captured, or measured frequencies of one or more reference materials.Signal generator 410 may be implemented as a multi-frequency signalgenerator or as multiple single-frequency signal generators. Broadcastantenna box 420 may comprise antennae 422 and 424 that work together.Antenna 422 may be implemented as a broadband antenna or as multiplesingle-frequency antennae, which receive(s) resonant frequencyinformation from signal generator 410. Antenna 424 may be implemented asa broadband antenna, or as multiple single-frequency antennae forbroadcasting resonance generating electromagnetic fields onto subject140. Antenna 424 may simultaneous broadcast multiple resonantfrequencies onto subject 140, which may impact target cells moreeffectively than other approaches such as transmitting differentsingle-frequency signals consecutively. Feedback antenna provides afeedback signal back to antenna 424. A feedback loop may not be requiredwith sufficient amplification via increased gain from signal generator410 and/or via an external amplifier on the output of signal generator410.

FIG. 5 illustrates disease treatment system 500 according to an exampleembodiment, which is similar to disease treatment system 400 exceptamplifier 171. In a positive feedback loop, amplifier 171 may situatebetween feedback antenna 170 and antenna 524 to provide for necessaryamplification of signals. Therefore, amplifier 171 sometimes removes theneed for increased gain from signal generator 510 and/or externalamplifier on the output of signal generator 510.

According to some embodiments, disease treatment systems may performprolonged exposure of a subject carrying a disease that containsselected reference material(s) critical to its progression or survival.Exposure may be continuous or intermittent. Electromagnetic exposureinduces amplified resonance within that critical component and stops adisease condition. In an example embodiment, exposing a subject to anelectromagnetic field lasts continuously for a time period sufficient tocause death of cells or organisms related to the disease condition.Targeted electromagnetic radiation may be suitable for treating cancer,bacterial infections, viral infections, and other maladies.

FIG. 6 is a flowchart showing process 600 for administering to a subjecthaving or at risk of having a disease condition with resonance-basedelectromagnetic radiation, according to an example embodiment (e.g.,system 100 as shown in FIG. 1). Process 600 may start in action 610,where one or more reference materials (e.g., reference material 112)related to the disease condition is selected. A reference material maybe biological substances extracted from cells or other sources or may besynthetic substances (e.g., a synthesized peptide sequence or asynthetic DNA sequence). For example, the reference material may be keyto the function, progression, viability, proliferation, continuation,and/or survival of the disease condition. In action 620, one or moreresonant frequencies of reference materials are captured. In action 630,a resonance frequency signal (e.g., signal 120) is generated to carry atleast one of the captured resonant frequencies. In action 640, amodulated broadband signal (e.g., signal 152) may be generated by asignal generator and provided to a first broadband antenna. In action650, a booster signal (e.g., signal 154) comprising a single, lowfrequency waveform may be optionally generated by the signal generatorand provided to a second broadband antenna. In action 660, anelectromagnetic field (e.g., represented by signal 136 in FIG. 1) may beradiated or effectuated jointly based on the resonant frequency signal,the modulated broadband signal, and the booster signal (if present). Inaction 670, a subject (e.g., subject 140) having or at risk of having atarget disease may be administered to by exposing the subject to theresonance generating electromagnetic field to affect biologicalactivities of cells or organisms causing or associated with the diseasecondition.

As will be understood by those skilled in the art, various embodiments(including those involving additional steps) are contemplated in lightof process 600. For example, in an example embodiment, a modulatedbroadband signal may be simultaneously modulated, in a signal generator,by at least a first waveform at a first frequency and at least a secondwaveform at a second frequency. The frequencies may be selected based onthe application. For example the first frequency of the first waveformmay be about 1 Hz or about 4 Hz, while the second frequency of thesecond waveform may be less than 1 MHz, less than 100 KHz (e.g., 2.2KHz), etc.

Embodiments disclosed herein may treat a disease condition with targetedelectromagnetic radiation from a suitable apparatus. Process oftreatment may include selection and isolation of substances critical tothe progression, viability, proliferation, continuation, and/or survivalof a particular disease such as cancer, bacterial or viral infectionsand other maladies. A reference material may provide resonantfrequencies (including harmonics) specific to that material which occurwithin the transmission bandwidth. Measurement and capture of theresonant frequencies may be performed for transmission and amplificationof resonant frequency signals. Optionally, resonance may be amplifiedthrough a feedback loop, which may or may not include a source ofadditional amplification. An electromagnetic field developed in the nearfield zone of an antenna array may be tuned by means of the feedbackloop.

FIG. 7A illustrates an example frequency spectrum, where multipleresonant frequencies are captured using Bacillus Calmette-Guerin (BCG),an attenuated strain of Mycobacterium bovis, as a reference material.Here, amplitude of an electromagnetic field is measured with a spectrumanalyzer in peak hold mode during exposure of BCG cells through threecycles. The peak hold function fixes highest amplitude at everyfrequency during the scan in each selected bandwidth. The approximateareas of resonance can be better depicted by using SPAN32 software tosubtract background radiation from each of the scans of the three cyclesand graphing the results. Using this graphic as an example permits oneto better visualize the resonant frequency trends. Precise determinationof the applicable resonant frequencies may be obtained by anoscilloscope. As shown in FIG. 7A, the frequency spectrum of theelectromagnetic field displays amplification at several resonantfrequencies, such as those at about 3,000 MHz, 3,170 MHz, 3,330 MHz,3,370 MHz, 3,410 MHz, 3,450 MHz, and 3,500 MHz. Some of thesefrequencies may be harmonic frequencies. One of ordinary skill in theart would recognize that a resonant frequency may actually be arepresentative frequency around which the electromagnetic spectradisplays over time relatively higher amplitude and fluctuation than inthose areas outside the frequencies of resonance.

Embodiments disclosed herein may be applied for treating a wide varietyof disease conditions. An example cancer is Glioblastoma Multiforme(GBM), where selected reference materials may include hsa-miRNA-38;mutated alpha-kinase 2 gene; Hsp70 (70 kDa heat shock protein), CHI3L1(chitinase-3-like protein 1), and/or the GBM cells themselves. Similarto the approach taken with BCG cells, spectral scans of exposed GBMcells were taken with a spectrum analyzer in the peak hold mode. In thisparticular case, however, the experiments were conducted inside of aFaraday room, thereby eliminating most background radiation. In order todepict the approximate areas of resonance, the spectral values of thescans conducted during the first two hours of exposure were averaged. Bysubtracting that average from consecutive scans on either side of theaverage, the resulting graphs, shown both individually and in compositeform, permit visualization of the approximate areas of resonance relatedto the particular targets selected to treat GBM. Precise determinationof actual resonant frequencies may be obtained by an oscilloscope. Asshown in FIG. 7B, resonant frequencies for these combined selectedreference materials within the electromagnetic spectra show increasedamplification at certain frequencies such as about 2,795 MHz, 2,808 MHz,2,820 MHz, 2,855 MHz, 2,860 MHz, and 2,865 MHz. These experimentsproduced a 30% reduction in GBM U-87 MG (a particular strain of GBMcancer chosen for the study) cell growth compared to a control group,which was the particular strain of GBM cancer chosen for the study.

Embodiments disclosed herein may also be applied for treating bacterialinfections, such as Mycobacterium Tuberculosis (Mtb), where selectedbacterial reference materials may include Phosphatidylmyo-inositolMannosides (PIM), Arabinogalactan, Lipoarabinomannan (LAM), andAlpha-crystallin. Similarly resonant frequencies for these combinedselected reference materials within the electromagnetic spectradisplayed show increased amplification at various frequencies specificto the reference material. These in vitro experiments produced a 98%reduction in Mtb H37-RA bacterial cell growth compared to a controlgroup which was the particular strain of Mtb chosen for the study.

Embodiments disclosed herein may also be applied for treating viralinfections, such as Human Immunodeficiency Virus (HIV), where selectedviral reference materials may include gp120, gp41, gp160, Gagpolyprotein, Env protein, sequences of viral RNA, p24 protein, and/orpro-viral DNA. This in vitro experiment produced a 99% reduction inHIV-1 cell growth compared to a control group.

Embodiments disclosed herein may treat a wide variety of diseaseconditions, which are not exhaustively listed herein. For example, theefficacy of a device with respect to Glioblastoma may be appliedeffectively for other diseases such as neurodegenerative disorders (e.g.Niemann Pick).

More specific example embodiments based on experimental studies areillustrated in FIGS. 8-13 to show that non-invasive electromagneticfield technologies have a significant inhibitory effect on theproliferation of various cells, such as glioblastoma for treating GBM(type U-87 MG) cells in culture, which relate to the treatment of GBM.Experimental studies observe the cellular and molecular responses ofU-87 glioblastoma cells to the effects of utilizing a tunable,non-ionizing radiation technology that is unlikely to induce the seriousside effects commonly observed with chemotherapy. Broadband (RF or lowmicrowave) electromagnetic field is tuned by means of proprietaryoscillating waveforms (either 1 Hz or 4 Hz), selected referencematerials and a positive feedback loop. By targeting specific cancermolecules (e.g., oligonucleotides and proteins) that contribute tocancer cell proliferation in glioblastoma with this technology,continuous exposure of cells for 54 hours (h) resulted in the inhibitionof cell growth (e.g., by about 30%) with concurrent cell death(apoptosis). The effects of a treatment system on cells in culture didnot appear to act on molecular processes common to temozolomide ordocetaxol treatment, thus acting by novel non-cytotoxic mechanism. Thistechnology has the potential to be customized for individual tumors andmay contribute to the emerging strategy of personalized medicine.

Disclosed herein are embodiments of non-invasive technologies, whichemploy non-ionizing, self-tuned, electromagnetic (EM) radiations thatcan be added to therapeutic armamentarium for GBM, alone or incombination with standard therapies. In example embodiments, firstreference materials reflecting critical components of the target cellsmay be selected. The system then amplifies the resonant frequencies ofthe reference materials. These resonances are transmitted to the targetcells which alter the behavior of corresponding reference molecules intarget cells, inducing mitotic arrest and cell death.

A treatment system used in experiments may emit, for example, anultra-low intensity (−50 to −90 dBm), broadband (100 MHz-3.5 GHz)electromagnetic radiation. Using an array of reference antennae andtransceivers coupled to a waveform generator, a treatment systemproduces a non-propagating electromagnetic field in a near-field zone ofthe antennae. A field may be self-tuned by means of oscillatingwaveforms (e.g., at 1 Hz [slow] or 4 Hz [fast] or other frequency) tocapture a resonant frequency and harmonics of selected referencematerials. Prescribed reference material resonances may then beamplified within a positive feedback loop, leading to the disruption ofcellular homeostasis and induction of cell death. Targeted molecules maybe selected by identifying critical oncogenic or tumor suppressiveprocesses in tumors.

Reference materials may then be isolated, prepared, stabilized, and usedto target primary glioblastomas (as modeled in experiments furtheroutlined below). The resonance generating electromagnetic fieldstechnology can also be customized for individual tumors. Unlike ionizingradiation, some disclosed embodiments employ non-ionizing, ultra-lowpower electromagnetic radiation, which is not associated withsignificant side effects. Thus, the present disclosure offers a novelmethod of therapy with minimal side effects that will be part of theemerging personalized medicine approach to combating diseases.

FIG. 8 illustrates an experimental system according to some embodiments.In experiments, to minimize inadvertent exposure of laboratory personnelto the electromagnetic fields generated during these experiments, anapparatus may be placed in an electromagnetic interference/RF shieldingFaraday room (sized 3.2 m×2.5 m×2.5 m). This shielding provides anattenuation of 100 dB to electric fields and plane waves from 14 KHz to10 GHz and 50 dB to magnetic fields at 14 KHz rising to 90 dB at 200 KHz(tested in accordance with IEEE-STD-299).

Custom-designed culture boxes may be assembled entirely from acrylic toprevent interference with electromagnetic fields associated with metalshelves and other components. Temperature/humidity readings insideculture boxes may be recorded on a continuous basis (e.g., 60 secondintervals) using Ethernet data recorders, and incubator temperatures maybe controlled using individual circulating heaters for each incubator.Appropriate carbon dioxide (CO2) levels are maintained by a continuousflow (e.g., 2.8 standard cubic feet per hour) of a gas mixture (e.g., 5%CO2 and 95% air) via a rotometer. An identical experimental setup may beestablished in a second laboratory over 30 meters away from the Faradayroom to serve as an unexposed control for experiments.

Reference materials that may be selected include hsa-miRNA-381; mutatedalpha-kinase 2 gene; Hsp70 (70 kDa heat shock protein) and CHI3L1(chitinase-3-like protein 1). DNA and RNA oligonucleotides may besynthesized and purified by high performance liquid chromatography(HPLC). Oligonucleotides may be solubilized, their volumes reduced, andthen transferred to an appropriate container for stabilization.Hsa-miR-381-5p (MIMAT0022862) sequence is 5′-AGCGAGGUUGCCCUUUGUAUAU-3′.This microRNA is highly expressed in brain tumors and may play a majorrole in glioma progression by targeting and inactivating LRRC4(leucine-rich repeat C4), a tumor suppressor gene that is specificallyexpressed in brain. Targeting this microRNA in U-87 MG cells maysignificantly inhibit cell viability, proliferation, and upregulateexpression of tumor suppressor LRRC4.

A U-87 MG cell line may have a homozygous mutated alpha-kinase 2 gene(hg18:uc002lhj.2, uc002lhk.1) with a 15 bp section 5′-AGGACACATCAACTG-3′deleted from the gene on the coding strand. A DNA oligonucleotide5′-AGGGAGACTG^TTACCATTGC-3′ may contain ten bp of an alpha-kinase 2coding sequence flanking the deletion site. Recombinant proteins maysolubilize in a PBS buffer. A buffer may be exchanged, with volumereduced, and then transferred to an appropriate container forstabilization.

Heat shock proteins may be expressed in all cells and may be more highlyexpressed in cancer cells due to the protection needed from the stressof their high metabolic requirements. In U-87 MG, a human recombinantprotein interacts with ATF5, an activating transcription factor, andstabilizes the protein from degradation. In other cancers, a humanrecombinant protein may protect anti-apoptotic proteins whilesuppressing pro-apoptotic activity. Targeting a human recombinantprotein may sensitize U-87 MG to chemical and oxidative stress,radiation therapy, as well as reduce stability of certain survivalproteins. This reference material may also be stabilized.

Chitinase-3-like protein 1 is a secreted glycoprotein that isoverexpressed in glioblastomas, and is highly overexpressed in U-87 MGcells. This protein is pro-angiogenic, contributes to radio resistanceand tumor progression in glioblastomas. Targeting another humanrecombinant protein CHI3L1 may decrease proliferation and metastasis inU-87 MG cells. This reference material may also be stabilized.

To ensure proper operation of a disease treatment system, in someembodiments an electromagnetic field intensity within a Faraday Room maybe measured continuously using a spectrum analyzer with a frequencyrange between 9 kHz and 3.5 GHz via a higher order proprietary antenna.During initial system calibration, spectral readings, e.g., viaproprietary software, may be taken outside of the Faraday Room.Background radiation intensity outside of the Faraday Room typicallyranged between −60 and −90 dBm.

After sealing the room, the following baseline scans may be taken toconfirm that performance of the Faraday room is in good order. Electricfield strength inside the Faraday room with the equipment operating mayaverage approximately 0.003 V/m. This level is far below the guidelinesissued for safe exposure to the general public. An increase in fieldintensity of approximately 15 to 20 dBm above unshielded backgroundradiation can be measured across the full 3.5 GHz span. When softwareconfirms that a latest scan indicates greater than 90% correlation levelwith a prior immediate scan (adequate tuning), cultures containing U-87MG cells may be inserted into a treatment culture box. Field intensitycan be further increased by 15 to 20 dBm in areas of resonance. Thechanges in field intensity may confirm that a system is operatingproperly.

FIG. 9 illustrates an example comparison of three spectral fieldstrengths: (910) the spectra of unshielded background radiation; (920)the spectra once a field has been tuned for four selected macromoleculesbefore U-87 MG cultures are introduced to the field; and (930) thespectra after U-87 MG cultures are brought into the tuned field.

By continuously measuring the broadband spectra of an electromagneticfield, proper operation of a treatment system may be confirmed, whichalso facilitates observation and analysis of changes in areas showingenhanced resonance response and spectral correlation percentages duringthe entire 54 hour period of exposure for each iteration of study.

U87-MG cells may be grown in minimal essential media containing 10%fetal bovine serum (FBS) and 1 mM sodium pyruvate. U87-MG cells may beroutinely passaged weekly at a density of 5000 cells/cm2 with mediachanges every three or four days, and maintained in a humidified 5% CO2incubator at 37° C.

U-87 MG cells may be plated at a density of 4×10⁵ cells/100-mm dish. 24hours after seeding, plates may be randomized to the treatment culturebox inside the Faraday room and exposed to a resonance generating field,or to the control culture box outside the Faraday room, to serve as theunexposed control. Cells may be exposed to slow modulation (1 Hz) orfast modulation (4 Hz) resonance generating electromagnetic field for 54hours continuously. At the end of exposure, half of the dishes may beprocessed and those respective cells may be harvested by trypsinization,and an aliquot of live/unfixed cells may be used to determine cellnumber, PS externalization, and caspase-3/7 activity using a cellanalyzer as described below. Remaining cells may be fixed for cell cycleanalysis and DNA fragmentation analysis as described below. Controlcells may be cultured under identical conditions. Remaining dishes maybe maintained for a further 96 hour outside the Faraday room, in a CO2incubator, and then processed in a manner similar to that describedabove.

U-87 MG cells may be plated at a density of 8×10⁵ cells/150-mm dish. 48hours after plating, cells may be treated with vehicle control or 25 nMdocetaxel for 72 hours. At the end of treatment, cells may be harvestedby trypsinization, and an aliquot of live/unfixed cells may be used todetermine cell number, PS externalization, and caspase-3/7 activityusing a cell analyzer. Remaining cells may be fixed for cell cycleanalysis using a cell analyzer and DNA fragmentation analysis by BDLSRII Flow Cytometry as described below.

U-87 MG cells harvested by trypsinization may be pooled with media pluswashes and pelleted by centrifugation. The cells may be re-suspended incomplete media diluted 1:10 with PBS (1% FBS/PBS). For determination ofcount and viability, an aliquot of the cell suspension may be diluted1:10 with a count & viability reagent, which differentially stainsviable and non-viable cells based on their permeability to two DNAbinding dyes, and incubated for 5 minutes in the dark at roomtemperature according to manufacturer's protocol. For detection ofphosphatidylserine (PS) externalization, an aliquot of cell suspensionappropriately diluted in 1% FBS/PBS may be mixed 1:1 with an Annexin V &dead cell reagent and incubated for 20 minutes in the dark at roomtemperature according to manufacturer's protocol. The Annexin V & deadcell reagent utilizes Annexin V to detect PS on the external membrane ofapoptotic cells. The dead cell marker 7-AAD, which is normally excludedfrom healthy and early apoptotic cells, may be used as an indicator ofcell membrane integrity. To assay caspase-3/7 activity, an aliquot ofcell suspension appropriately diluted in 1% FBS/PBS may be mixed withcaspase-3/7 reagent and incubated for 30 minutes in the dark at 37° C.according to manufacturer's protocol. The caspase-3/7 reagent is a cellmembrane permeable DNA binding dye that is linked to DEVD peptidesubstrate.

Cleavage by active caspase-3/7 releases the dye within the cell andresults in translocation to the nucleus, binding to DNA and highfluorescence. For cell cycle analysis, an ethanol-fixed cell suspensionmay be obtained by taking an aliquot of cell suspension, pelleting bycentrifugation, resuspending in PBS, and fixing in 70% ethanol at −20°C. Fixed cells may be washed in PBS/0.2% BSA, pelleted bycentrifugation, resuspended in a cell cycle reagent, and incubated for30 minutes in the dark at room temperature according to manufacturer'sprotocol. A cell cycle reagent may be a proprietary formulationcontaining the DNA intercalating dye propidium iodide and RNAse A. Themultiparametric fluorescent detection of individual cells may beperformed by a cell analyzer, a microcapillary flow cytometer equippedwith a 532-nm laser, a forward scatter and two fluorescence (YLW 576/26,RED 680/30) detectors. Data acquisition and analysis of count &viability, Annexin V & dead cell, and caspase-3/7 assays may be done byanalysis software. Post-acquisition analysis of cell cycle data may beperformed using software (e.g., FlowJo 7.6.5).

For analysis of DNA fragmentation, U-87 MG cells may be harvested bytrypsinization, collected by centrifugation, fixed in 2% formaldehyde inPBS, and permeabilized in 70% EtOH at −20° C. DNA strand breaks in cellsundergoing apoptosis may be indirectly labeled with bromodeoxyuridine byterminal transferase and detected by FITC-conjugated monoclonal antibodyto bromodeoxyuridine using the APO-BRDU kit according to manufacturer'sprotocol. Cells may be counterstained with 5 μg/ml propidium iodidecontaining RNase A for detection of total DNA, and two-color analysis ofDNA strand breaks and cell cycle may be achieved by flow cytometry. Flowcytometric analysis may be performed on a BD LSR II Flow Cytometerequipped with three excitation lasers (405-nm, 488-nm and 633-nm). ForDNA fragmentation analysis, FITC and PI signals may be collected by488-nm octagon detection array with no color compensation. 10,000 eventsgated on single cell populations may be collected and post-acquisitionanalysis may be performed using software FlowJo 7.6.5.

FIGS. 10A-10E illustrate comparisons of changes in control cells versuscells exposed at slow modulation for the U-87 MG strain of GBM.Specifically, FIG. 10A shows changes in U-87 MG cell number; FIG. 10Bshows changes in U-87 MG cell cycle, including the sub G0 population;FIG. 10C shows changes in DNA fragmentation in U-87 MG in all fourquadrants of the histogram; FIG. 10D shows changes in caspase-3/7activation; and FIG. 10E shows changes in phosphotidylserine (PS) asmeasured by Annexin V staining. Data are represented as Mean±SD of threeindependent biological experiments (*p<0.1; ns—not significant).

After 54 hours' exposure to a resonance generating field, there may be asignificant decrease of 30% in U-87 MG cell number (1.0×10⁶ cells/dish),relative to unexposed control cells (1.5×10⁶ cells/dish), as shown inFIG. 10A. After the cells are removed from the irradiated area, cellgrowth rate may be restored, although at 96 hours' post exposure, thecell number of exposed cells (6.3×106 cells/dish) still lag 31% behindthat of the unexposed cells (9.1×106 cells/dish). These imply thatresonance generating electromagnetic fields, tuned to the fourreferences materials outlined above, may arrest cell growth as long asthe cells are in the field. Cell growth may be restored in the absenceof continued exposure.

Exposure of U-87 MG cells to slow modulation resonance generatingelectromagnetic fields also induces a significant and apparentlysustained change in cell cycle kinetics compared to unexposed controls,as shown in FIG. 10B and Table 1.

Table 1 shows cell cycle analysis of U-87 MG cells after exposure toresonance generating electromagnetic fields at slow modulation. At theend of 54 hours exposure or 96 hours post exposure, cells may beharvested and cell cycle may be determined by incubating the cells in acell cycle reagent, following the manufacturer's protocol. Datarepresent the Mean±SD of three independent biological replicates(*p<0.1).

TABLE 1 Sub-G₀ G1 S G2/M 54 hour exposure unexposed 0.5 ± 0.4 51.5 ±1.0  19.7 ± 1.0 28.5 ± 1.3  exposed 0.6 ± 0.6 49.7 ± 0.4* 16.8 ± 2.433.1 ± 2.5* 96 hour post exposure unexposed 0.7 ± 0.7 59.7 ± 4.7  14.4 ±3.5 24.7 ± 1.7  exposed 0.8 ± 0.4 52.7 ± 2.4* 18.3 ± 2.9 27.6 ± 1.3*

A decrease in cell number shown in FIG. 10A may be accompanied by asignificant decrease in the proportion of the cells in the G1 phase ofthe cell cycle and a concomitant significant increase in the proportionof cells in G2/M. Redistribution of cells in a cell cycle may bemaintained even in cells 96 hours post-exposure, suggesting thatresonance generating electromagnetic fields under slow modulation havean extended effect on cell cycle kinetics, even though cell numberincreases once cells are removed from the field. This somewhatparadoxical effect may be explained by the induction of cell death, asmeasured by DNA fragmentation, as shown in FIG. 10C and Table 2, whichshow that resonance generating electromagnetic fields induce a largeincrease in TUNEL positive cells after 54 hours of exposure. 96 hourspost exposure, DNA fragmentation may have returned to the levels seen inunexposed cells.

Table 2 shows analysis of DNA fragmentation of U-87 MG cells afterexposure to resonance generating electromagnetic fields at slowmodulation. At the end of 54 hour exposure or 96 hour post exposure,cells may be harvested, fixed in PBS, and permeabilized. DNAfragmentation may be determined by flow cytometry. Data show percentageof the cell population with DNA fragmentation in all four quadrants ofthe histogram and represent the Mean±SD of three independent biologicalreplicates (*p<0.1).

TABLE 2 DNA Frag DNA Frag DNA Frag DNA Frag (+) (+) (−) (−) ApoptoticCells Cells Apoptotic bodies (upper (lower bodies (upper left rightright (lower left quadrant) quadrant) quadrant) quadrant) 54 hourexposure unexposed 0.03 ± 0.01 0.97 ± 0.41 98.5 ± 0.25 0.53 ± 0.28exposed 0.05 ± 0.03 4.09 ± 2.79 95.5 ± 3.0  0.39 ± 0.16 96 hour postexposure unexposed 0.03 ± 0.01 0.41 ± 0.29 98.7 ± 0.03 0.81 ± 0.29exposed 0.07 ± 0.04 1.14 ± 0.86 98.0 ± 0.74 0.81 ± 0.17

Analysis of apoptosis using two additional markers ofapoptosis-caspase-3/7 activation (FIG. 10D), a marker of the earlystages of apoptosis, and Annexin V staining of phosphytidylserine on theouter leaflet of the dying cell and a marker of the later stages ofapoptosis (FIG. 10E), suggest that neither of common apoptotic processesis involved in the induction of cell death by resonance generatingelectromagnetic fields. It is also possible that the apoptotic eventsare sufficiently asynchronous that the population of cells expressingthese markers never reach significance relative to the background levelsof cell death in the control cultures.

U-87 MG cells in separate experiments may be exposed to a resonancegenerating electromagnetic field for 54 hours continuously at fastmodulation, the results from which are shown in FIG. 11, Table 3, andTable 4.

FIGS. 11A-11E represent an analysis of U-87 MG cells proliferation andapoptosis after exposure to resonance generating electromagnetic fieldsat fast modulation. FIGS. 11 A-E show comparisons of changes in controlcells versus exposed cells. Specifically, FIG. 11A shows changes in U-87MG cell number; FIG. 11B shows changes in U-87 MG cell cycle, includingthe sub G0 population; FIG. 11C shows changes in DNA fragmentation inU-87 MG in all four quadrants of the histogram; FIG. 11A shows changesin caspase-3/7 activation; and FIG. 11A shows changes in PS as measuredby Annexin V staining. Data are represented as Mean±SD of fourindependent biological experiments (*p<0.1; ns—not significant).

Table 3 is a cell cycle analysis of U-87 MG cells after exposure toresonance generating electromagnetic fields at fast modulation. At theend of 54 hour exposure or 96 hour post exposure, cells may be harvestedand cell cycle may be determined by incubating the cells in a cell cyclereagent, following the manufacturer's protocol. Data represent themean±SD of four independent biological replicates (*p<0.1).

TABLE 3 Sub-G₀ G1 S G2/M 54 hour exposure unexposed 0.2 ± 0.3  52.9 ±3.5 16.3 ± 4.1 30.7 ± 7.3 exposed 0.6 ± 0.3* 49.4 ± 3.9 16.9 ± 2.4 32.8± 5.1 96 hour post exposure unexposed 0.5 ± 0.5 55.1 ± 5.8 15.6 ± 3.528.5 ± 7.4 exposed 0.4 ± 0.3 52.8 ± 3.8 16.7 ± 4.2 29.5 ± 5.6

Table 4 is an analysis of DNA fragmentation of U-87 MG cells after toresonance generating electromagnetic fields at fast modulation. At theend of 54 hour exposure or 96 hour post exposure, cells may beharvested, fixed in PBS, and permeabilized. DNA fragmentation may bedetermined by flow cytometry. Data show percentage of the cellpopulation with DNA fragmentation in all four quadrants of the histogramand represent the mean±SD of four independent biological replicates(*p<0.1).

TABLE 4 DNA Frag DNA Frag DNA Frag DNA Frag (+) (+) (−) (−) ApoptoticCells Cells Apoptotic bodies (upper (lower bodies (upper left rightright (lower left quadrant) quadrant) quadrant) quadrant) 54 hourexposure unexposed 0.02 ± 0.03  0.6 ± 0.29 99.2 ± 0.34  0.24 ± 0.07exposed 0.04 ± 0.02  3.99 ± 1.76* 95.7 ± 1.73* 0.28 ± 0.06 96 hour postexposure unexposed 0.03 ± 0.02 1.26 ± 0.84 98.1 ± 0.89 0.65 ± 0.30exposed 0.04 ± 0.05 2.01 ± 1.93 97.3 ± 1.96 0.64 ± 0.31

To compare resonance generating electromagnetic fields with the wellcharacterized effects of chemotherapeutic drugs, U87 cells may betreated with 25 nM docetaxel for 72 hours. At this dose andconcentration docetaxel has been shown to be as effective astemozolomide in arresting cell growth in U-87 MG cells. Docetaxeldecreases U-87 MG cell number by approximately 20%, although this effectdoes not reach significance over the 72 h time course. This effect isaccompanied by a significant decrease in the proportion of the cells inG1 along with significant increases in the proportion of cells in Sphase and also in the sub G0 population, as shown in FIG. 12B and Table5. The increase in the sub G0 population does not appear to beaccompanied by a significant increase in DNA fragmentation, as shown inFIG. 12D and Table 5), but is associated with a significant increase incaspase-3/7 activation and Annexin V positive cells, as shown in FIGS.12D-E.

FIGS. 12A-12E are an analysis of U-87 MG cells proliferation andapoptosis after 72 hour treatment with 25 nM docetaxel. FIGS. 12 A-Eshow comparisons of changes in U-87 MG cell number in the absence andpresence of 25 nM docetaxel. Specifically, FIG. 12A shows changes inU-87 MG cell number; FIG. 12B shows changes in U-87 MG cell cycle,including the sub G0 population; FIG. 12C shows changes in DNAfragmentation in U-87 MG in all four quadrants of the histogram; FIG.12D shows changes in caspase-3/7 activation; and FIG. 12B shows changesin PS as measured by Annexin V staining. Data are represented as Mean±SDof three independent biological experiments (*p<0.1; ns—not significant)with the exception of DNA Fragmentation which had only two independentbiological experiments.

Table 5 is a cell cycle analysis of U-87 MG cells after 72 hourtreatment with 25 nM docetaxel. At the end of 72 hour treatment, cellsmay be harvested and cell cycle may be determined by incubating thecells in a cell cycle reagent and following the manufacturer's protocol.Data represent the mean±SD of three independent biological replicates(*p<0.1).

TABLE 5 72 hour treatment Sub-G₀ G1 S G2/M DMSO 0.6 ± 0.3  53.0 ± 5.8 14.2 ± 2.4  31.8 ± 7.1 Docetaxel 9.9 ± 3.9* 39.7 ± 4.8* 26.7 ± 1.7* 22.1± 4.4

Table 6 is an analysis of DNA fragmentation of U-87 MG cells after 72hour treatment with 25 nM docetaxel. DNA fragmentation may be determinedby flow cytometry as described above. Data show percentage of the cellpopulation with DNA fragmentation in all four quadrants of the histogramand represent the Mean±SD of two independent biological replicates(*p<0.1).

TABLE 6 72 hour treatment DNA Frag DNA Frag DNA Frag DNA Frag (+) (+)(−) (−) Apoptotic Cells Cells Apoptotic bodies (upper (lower bodies(upper left right right (lower left quadrant) quadrant) quadrant)quadrant) DMSO 0.06 ± 0.06 1.70 ± 1.70 97.6 ± 2.12 0.72 ± 0.39 Docetaxel0.10 ± 0.0  2.11 ± 1.69 94.2 ± 0.07 3.57 ± 1.65

Test data presented herein demonstrates that resonance generatingelectromagnetic fields may reduce cell growth, alter cell cyclekinetics, and increase DNA fragmentation in U-87 MG cells. Whileresonance generating electromagnetic fields at slow modulation may showan increase (e.g., significant increase) in DNA fragmentation; neitherAnnexin V staining nor caspase-3/7 activation in U-87 MG cells seemscorrelated to the induction of apoptosis. Exposure of U-87 MG cells toresonance generating electromagnetic fields at fast modulation maydecrease total cell number by 30%, similar to slow modulation; however,at fast modulation, there may be an increase (e.g., significantincrease) in the sub G0 population (indicative of the formation ofapoptotic bodies) and DNA fragmentation. Data suggests that resonantelectromagnetic fields, at both slow and fast modulation, induce DNAfragmentation and cell death by a novel mechanism that does not requirecaspase-3/7 activation, or exposure of PS on the outer leaflet of thedying cell. Treatment of U87-MG cells with 25 nM Docetaxel for 72 hoursmay induce a 20% decrease in cell number and an increase (e.g.,significant increase) in the sub G0 population, and caspase 3/7activation. Along with increases in PS exposure as measured by AnnexinV, classical apoptotic pathways are present in these cells and can beactivated with the appropriate stimulus. Both resonance generatingelectromagnetic fields and docetaxel appear to induce G2/M arrestfollowed by cell death. Because the induction of mitotic cell deathafter treatment with docetaxel induces caspase-3/7, while resonancegenerating electromagnetic fields do not, intracellular mechanismsmediating resonance generating electromagnetic fields induced DNAfragmentation may be different from pathways utilized by both docetaxeland temozolomide that show equivalent results in cell culture.Comparison of the effects of resonance generating electromagnetic fieldsand docetaxel on U-87 MG cell number shows that resonance generatingelectromagnetic fields at both slow and fast modulation may be as goodas, or better than, docetaxel monotherapy in culture.

Unlike conventional electromagnetic treatments, the use of referencematerials may capture one or more exact resonant frequencies andharmonics of a target without having to calculate the appropriateresonant frequency or determine an effective resonant frequency for aparticular outcome by trial-and-error. In addition, unlike TTFields,there are unlikely to be unwanted dermatologic side effects. Someembodiments herein may use four different macromolecular targets asreference materials, which may be equally effective, or some may be moreeffective than others. The effects of targets may be additive orsynergistic.

Targets may be optimized based on genome wide comparisons of the commongenetic lesions among glioblastoma tumors. Optimization may identify oneor more combinations of reference materials for treatment of specificsubsets of tumors. For example, tumors with IDH mutations may betargeted more effectively with reference materials based on known IDHmutations, while tumors known to harbor other mutations may not beaffected. The effects of resonance generating electromagnetic fieldsdescribed herein, with the possible exception of cell cycle kineticsafter exposure to slow modulation, may be reversed in surviving cellswhen they are no longer exposed to a field. Thus, the present disclosuremay determine whether patients with GBM will require longer term,intermittent, or continual exposure to resonance generatingelectromagnetic fields to stop growth and progression of GBM. There-initiation of cell growth noted when the cells are removed from thefield may relate to the enrichment of glioblastoma stem-like cells amongthe surviving population. These stem-like cells may be effectivelytargeted concurrently using reference antennae with OLIG2 and SALL2 astargets to self-tune the resonance generating electromagnetic fields toreduce or prevent the initiation of cell growth.

Given the advantage of inexpensive whole genome sequencing analysis, onemay obtain individualized deep sequencing data derived from the tumor.This would permit the selection of patient specific targets for thereference antennae, which could be used for second line therapy incombination with other modalities. Xenograft studies may be used todetermine whether control of GBM tumor growth can be achieved withintermittent exposure to resonance generating electromagnetic fields.The technology disclosed herein induces a significant inhibitory effecton U-87 MG, and is unlikely to induce toxic or other negative sideeffects. Thus, the possibility of repetitive therapy sessions usingresonance generating electromagnetic fields may create valuable medicaloptions and a paradigm shift in cancer therapy.

Further Details on the Effect of Resonance Generating ElectromagneticFields

Resonance generating electromagnetic fields disclosed herein bring aboutunique effects. Some electromagnetic fields may give rise to ioncyclotron resonance in two ways when charged particles are moving withina uniform magnetic field: (i) setting an AC peak intensity equal to astatic magnetic field; and (ii) when a linearly polarized oscillatingelectric field is aligned 90° to a static magnetic field. In eithercase, resonance for a particular ion (e.g., Ca2+, K+) can occur at thesame modulation frequency given an equivalent angular velocity and anappropriate, but different field strength. In an RF/low microwavefrequency range, the spatial period of an electromagnetic wave emittedby a conventional antenna is much longer than typical molecular sizes.This means that the amplitude may be the same for any part of themolecule, that is, a molecule interacts with spatially homogeneouselectric and magnetic fields but varying in time. Electrodynamics saysthat such an electric field results in rotation of molecules that have afinite dipole moment.

The present disclosure uses, according to some embodiments,multi-solenoidal antennae that are distinct from conventional dipoleantennae to produce resonance generating electromagnetic fields. Agenerated field is proportional to the second derivative of an electriccurrent. Multi-solenoidal antennae may produce even static-like electricand/or magnetic fields, which are proportional to the third andhigher-order time derivatives of a current.

Modulation of electromagnetic fields may excite cyclotron and otherresonances. Prolonged exposure to amplified resonances may produce ameasurable effect, such as a change in spectra and/or biologicalactivity.

FIG. 13 illustrates a change in spectra from cultures exposed for twohours (represented by curve 1310) and the same cultures after beingexposed for 18 hours (represented by curve 1320). Specifically, FIG. 13shows spectra in a 350 MHz area of interest resulting from U-87 MG cellsexposed to resonance generating electromagnetic fields tuned for four,U-87 MG macromolecules taken inside an RF/EMI shield room.

Resonance generating electromagnetic fields disclosed herein may haveseveral useful elements, e.g., (i) antennae that transmit/receive RF/lowband microwave radiation in an ultra-wide spectral range; (ii) theproperties of disclosed RF/low microwave are distinct from what isradiated by conventional antennae and also those of noise, and theseproperties make it possible to excite electromagnetic resonances thatcannot be excited by conventional RF waves/microwaves; and, (iii) anoptional continuous, self-tuning, positive feedback circuit thatincludes reference material targets specific to the biological substancebeing treated. These characteristics may enable accurate tuning ofresonance generating electromagnetic fields that are otherwiseunavailable for various reasons. For example, ion cyclotron resonance isrelated to field strength and angular velocity of the waveform. As acase in point, separate experiments on bone growth demonstrate that, forthe same modulation frequency (16 Hz) and different field intensities(20.9 μT and 40.7 μT), first inducing ion cyclotron resonance of Ca2+increases bone growth and second inducing ion cyclotron resonance of K+inhibits bone growth. For another example, ion cyclotron resonance mayalso occur at higher harmonics of a fundamental resonance frequency.Because resonance generating electromagnetic fields may be generatedwithin a broadband frequency range, harmonic resonance may enhance theeffect of these fields on target(s). For yet another example, unlikeconventional monochromatic electromagnetic fields without a feedbackloop that may be affected by field strength variations, damping andother factors herein give resonance generating electromagnetic fieldsadvantages in application.

As will be understood by those skilled in the art who have the benefitof the instant disclosure, other equivalent or alternative compositions,devices, methods, and systems for resonance-based disease treatment canbe envisioned without departing from the description contained herein.Accordingly, the manner of carrying out the disclosure as shown anddescribed is to be construed as illustrative only.

Persons skilled in the art may make various changes in the nature,number, and/or arrangement of parts or steps without departing from thescope of the instant disclosure. For example, the size of a deviceand/or system may be scaled up or down to suit the needs and/or desiresof a practitioner. Each disclosed method and method step may beperformed in association with any other disclosed method or method stepand in any order according to some embodiments. Where the verb “may” or“can” appears, it is intended to convey an optional and/or permissivecondition, but its use is not intended to suggest any lack ofoperability unless otherwise indicated. Where open terms such as“having” or “comprising” are used, one of ordinary skill in the arthaving the benefit of the instant disclosure will appreciate that thedisclosed features or steps optionally may be combined with additionalfeatures or steps. Where “based on” or “based upon” is used, one ofordinary skill in the art having the benefit of the instant disclosurewill appreciate that it means one thing is dependent at least in part onanother thing, directly or indirectly, exclusively or non-exclusively.Such option may not be exercised and, indeed, in some embodiments,disclosed systems, compositions, apparatuses, and/or methods may excludeany other features or steps beyond those disclosed herein. Elements,compositions, devices, systems, methods, and method steps not recitedmay be included or excluded as desired or required. Persons skilled inthe art may make various changes in methods of preparing and using acomposition, device, and/or system of the disclosure.

Also, where ranges have been provided, the disclosed endpoints may betreated as exact and/or approximations as desired or demanded by theparticular embodiment. Where the endpoints are approximate, the degreeof flexibility may vary in proportion to the order of magnitude of therange. For example, on one hand, a range endpoint of about 50 in thecontext of a range of about 5 to about 50 may include 50.5, but not 52.5or 55 and, on the other hand, a range endpoint of about 50 in thecontext of a range of about 0.5 to about 50 may include 55, but not 60or 75. In addition, it may be desirable, in some embodiments, to mix andmatch range endpoints. Also, in some embodiments, each figure disclosed(e.g., in one or more of the examples, tables, and/or drawings) may formthe basis of a range (e.g., depicted value +/−about 10%, depicted value+/−about 50%, depicted value +/−about 100%) and/or a range endpoint.With respect to the former, a value of 50 depicted in an example, table,and/or drawing may form the basis of a range of, for example, about 45to about 55, about 25 to about 100, and/or about 0 to about 100.

All or a portion of a device and/or system for disease treatment may beconfigured and arranged to be disposable, serviceable, interchangeable,and/or replaceable. These equivalents and alternatives along withobvious changes and modifications are intended to be included within thescope of the present disclosure. Accordingly, the foregoing disclosureis intended to be illustrative, but not limiting, of the scope of thedisclosure as illustrated by the appended claims.

Headings (e.g., Title, Abstract, Background, Detailed Description) areprovided in compliance with regulations and/or for the convenience ofthe reader. They do not include and should not be read to includedefinitive or over-arching admissions as to the scope and content ofprior art or limitations applicable to all disclosed embodiments.

What is claimed is:
 1. A method for administering to a subject having orat risk of having a disease condition resonance-based electromagneticradiation, the method comprising: selecting one or more referencematerials related to the disease condition; capturing one or moreresonant frequencies of the reference materials; generating a resonantfrequency signal comprising at least one of the captured resonantfrequencies; generating a modulated broadband signal that issimultaneously modulated, in a signal generator, by a first waveform ata first frequency and at least a second waveform at a second frequency;radiating an electromagnetic field based on the resonant frequencysignal; and exposing the subject to the electromagnetic field to affectbiological activities of cells related to the disease condition, whereinthe electromagnetic field is effectuated based on both the resonantfrequency signal and the modulated broadband signal.
 2. The method ofclaim 1, further comprising generating, by the signal generator, abooster signal comprising a single, low frequency waveform having afrequency lower than 100 KHz, wherein the electromagnetic field iseffectuated further based on the booster signal, and wherein the firstfrequency of the first waveform is substantially identical with the lowfrequency of the booster signal but different from the second frequencyof the second waveform.
 3. The method of claim 2, wherein the modulatedbroadband signal is received by a first broadband antenna via cable,wherein the booster signal and the resonant frequency signal arereceived by a second broadband antenna via cables, and wherein theelectromagnetic field used to expose the subject is effectuated based onradiations from both the first and second broadband antennae, which arecoupled in an antenna box.
 4. The method of claim 2, wherein, themodulated broadband signal has a bandwidth of 1 MHz or greater, andwherein the first frequency of the first waveform is about 1 Hz or about4 Hz, whereas the second frequency of the second waveform is less than 1MHz.
 5. A method for administering to a subject having or at risk ofhaving a disease condition resonance-based electromagnetic radiation,the method comprising: selecting one or more first reference materialsrelated to the disease condition; capturing one or more first resonantfrequencies of the reference materials; generating a first resonantfrequency signal comprising at least one of the captured resonantfrequencies; radiating a first electromagnetic field based on the firstresonant frequency signal; and exposing the subject to the firstelectromagnetic field to affect biological activities of cells relatedto the disease condition, wherein the one or more reference materialsare critical to the function, progression, viability, and/orcontinuation of a cell or organism causing or associated with thedisease condition, and wherein the resonant frequency signal comprisesat least two captured resonant frequencies, including any harmonics thatare used simultaneously to expose the subject.
 6. The method of claim 5,further comprising: selecting one or more second reference materialsrelated to the disease condition; capturing one or more second resonantfrequencies of the second reference materials; generating a secondresonant frequency signal comprising at least one of the captured secondresonant frequencies; radiating a second electromagnetic field based onthe second resonant frequency signal; and exposing the subject to thesecond electromagnetic field simultaneously with exposure to theelectromagnetic field.
 7. The method of claim 1, further comprisingtuning frequencies of the electromagnetic field via a feedback loop tomaximize resonance of the reference materials.
 8. The method of claim 1,further comprising, after selecting the reference materials but beforecapturing the one or more resonant frequencies, placing and isolatingthe reference materials inside a shielded container, wherein theexposure of the subject to the electromagnetic field lasts for asufficient cumulative period of time to cause death of cells related tothe disease condition or slow the progression of the disease condition.9. The method of claim 5, wherein the disease condition relates toGlioblastoma Multiforme (GBM), and wherein the one or more referencematerials are selected from the group consisting of hsa-miRNA-38,mutated alpha-kinase 2 gene, Hsp70 (70 kDa heat shock protein), CHI3L1(chitinase-3-like protein 1), and GBM cells.
 10. An electromagneticresonance-based disease treatment system comprising: a processing unitconfigured to generate a resonant frequency signal that carries at leastone frequency at which reference materials related to a diseasecondition resonate; and a radiating antenna configured to radiate anelectromagnetic field based on the resonant frequency signal, whereinthe electromagnetic field is operable on a subject having or at risk ofhaving the disease condition by exposing the subject to theelectromagnetic field in order to treat the disease condition, whereinthe processing unit comprises a signal generator configured to generatea modulated broadband signal at least via simultaneously modulation by afirst waveform at a first frequency and at least a second waveform at asecond frequency, wherein the electromagnetic field is effectuated basedon both the resonant frequency signal and the modulated broadbandsignal, wherein the signal generator is further configured to generate abooster signal comprising a single, low frequency waveform below about100 KHz, wherein the electromagnetic field is further based on thebooster signal, and wherein the first frequency of the first waveform issubstantially identical with the low frequency of the booster signal butdifferent from the second frequency of the second waveform.
 11. Thesystem of claim 10, wherein the resonant frequency signal carries aplurality of resonant frequencies that are used simultaneously to exposethe subject.
 12. The system of claim 11, wherein the processing unitcomprises a multi-frequency signal generator or multiple singlefrequency signal generators configured to generate the plurality ofresonant frequencies based on calculated, digitized, or measuredresonant frequencies of the reference materials.
 13. The system of claim10, further comprising: a feedback antenna located in a near field zoneof the subject; and an amplifier coupled to the feedback antenna and theradiating antenna, wherein the feedback antenna, the amplifier, and theradiating antenna form a feedback loop operable to tune frequencies ofthe electromagnetic field.
 14. A system for administering to a subject aresonance-based electromagnetic radiation, the system comprising: meansfor selecting one or more target substances relevant to a diseasecondition; and means for determining at least one resonant frequency atwhich the target substances resonate; means for generating a broadbandsignal that is simultaneously modulated at two frequencies of twodifferent waveforms; means for generating a resonant frequency signalcomprising the at least one resonant frequency; means for radiating anelectromagnetic field based on the broadband signal and the resonantfrequency signal; and means for exposing a subject carrying the diseasecondition to the electromagnetic field to affect biological activitiesof cells relevant to the disease condition.